Antibodies have been prepared to several membrane proteins of B. subtilis that may be involved in secretion. These antibodies will be used to try to inhibit protein secretion into vesicles in vitro, and to quantitate and prepare the antigens. Another approach being used to try to identify the machinery of secretion is crosslinking of membrane proteins to ribosomes or to nascent chains. Subunit A of cholera toxin and exotoxin A of Pseudomonas aeruginosa have been shown to be secreted posttranslationally and to be synthesized as larger precursors. In membrane-bound penicillinase and in several other lipoproteins of B. licheniformis the lipid is shown to be attached during chain growth. The lipid has been shown by others to be a glyceride linked as a thioether to cysteine, rather than a phospholipid. We plan to try to identify sites of attachment of the toxins to inner and to outer membrane during secretion, and to study the kinetics of incorporation of lipid into growing chains of lipoproteins.